DNA damage response of A549 cells treated with particulate matter (PM10) of urban air pollutants
Introduction
There is growing concern that exposure to airborne particulate matter (PM) is associated with an increase in morbidity and mortality and that it could augment the risk of lung cancer [1], [2], [3]. PM of an aerodynamic diameter ⩽10 mm (PM10) consists of dust, soot, and other solid, liquid, and aerosol particles, as well as its chemical constituents. Although the mechanisms of PM10-related health effects remain incompletely studied, there is increasing evidence that exposure to PM10 has a major effect in several experimental models [4], [5]. In addition, some of these changes are strongly related to an increase in reactive oxygen species (ROS) [6], [7]. In this regard, one of the most important changes induced by PM10 is DNA damage [8], [9] and the level of this damage has been correlated with mutagenicity and tumor formation in experimental models [10], [11]. The induced DNA damage by PM10 and its constituents involves the formation of DNA double-strand breaks (DSBs) [8], [9], [12], [13]. In response to DSBs caused by other agents like ionizing radiation [14], the histone H2A.X undergoes phosphorylation at Ser139 [14]. The phosphorylated H2A.X protein is defined as γH2A.X and this phosphorylation is mediated by phosphoinositide 3-kinase-related protein kinases (PIKKs), including ataxia telangiectasia mutated (ATM), ATM and Rad-3 related kinase (ATR), and DNA dependent protein kinase (DNA-PKcs) [15]. ATM and ATR, in turn, activate the checkpoint kinases, Chk1 and Chk2, and tumor suppressor 53-binding protein 1 (53BP1). 53BP1 binds to the DNA-binding domain of p53, enhances p53-mediated transcriptional activation, and rapidly colocalizes with γH2A.X in response to DNA damage induced by ionizing radiation [16]. In addition, Chk1 and Chk2 kinases have several effectors, including cell division 25 (Cdc25) family proteins, which have phosphatase activity related to cell cycle regulation, specifically Cdc25A, in response to ionizing radiation [17]. On the other hand, after DNA damage, p53 initiates different transcriptional programs that lead to cell cycle arrest, cellular senescence, or apoptosis in response to γ-radiation through the ATM/ATR and Chk1/Chk2 kinases [18]. To this regard, before p53 induction, a disruption between the complex formed by p53 and MDM2, a p53-specific E3 ubiquitin ligase, is needed for its biological response [19]. In the setting of DNA damage, the cell undergoes cell cycle arrest to provide time to carry out DNA repair [9], [13] or apoptosis, depending on the PM10 concentration used. Based on this information, we decided to investigate if the above cell-signaling pathway (ATM/ATR/Chk1/Chk2/p53) is involved in the response to the DNA damage induced by PM10 exposure in epithelial lung cancer cells even when cells are exposed to a sub-lethal concentration. Since there is an increase in DNA damage correlated to the oxidative stress induced by environmental pollutants [20], [21], we decided to use trolox, a hydroxyl radical scavenger, to investigate if a connection exists between the effect of PM10 exposure on DNA damage and if this effect is related to ROS formation. This study evidences that PM10 activates sensors (53BP1), transducers (ATM/ATR), and effectors (H2A.X, Chk1, Chk2, p53, and MDM2) of DNA damage, pointing toward a strong relation between DNA damage and oxidative stress. Even though, this signal transduction pathway is activated in response to DNA damage after 24 h, a senescence-like phenotype is observed in cells exposed to PM10 after 48 h. These findings provide important clues on the role of PM10 exposure in DNA damage and senescence-like events that are strongly involved in cancer development.
Section snippets
PM10 sampling
PM10 was collected in a commercial zone (with major traffic sources) of Mexico City using a high-volume particle collector with a flux of 1.13 m3/min (GMW model 1200 VFC HVPM10; Sierra Andersen, Smyrna, GA, USA). PM10 was collected on 3.0-μm pore size cellulose nitrate filters (Sartorius AG, Goettingen, Germany), 3 days a week from October 2004–May 2005. Filters were maintained in the dark at 4 °C in a desiccator prior to particle removal. Particles were gently scraped off the membranes with a
PM10 exposure induces DNA damage in A549 cells
To investigate the effect of PM10 in human A549 cells, we first examined the ability of PM10 to induce DNA damage using the comet assay; a well-established method for the DNA damaged detection. In this assay, the damaged DNA migrates out of the nucleus forming a tail, which can be quantified using image analysis. Fig. 1 shows that cells exposed to 10 μg/cm2 of PM10 after 24 h have an increased tail length compared to control cells (p < 0.001). Trolox treatment of cells exposed to PM10-induced a
Discussion
A549 cells were exposed to PM10 using a concentration of 10 μg/cm2 and the effect of 10 μM trolox, a hydroxyl radical scavenger, was explored to investigate if the DNA damage induced by PM10 is mediated by oxidative stress. One of the most important characteristics of PM10 is their ability to penetrate into the thoracic part of the airways, where they could have adverse effects [33]. The effects observed in this work could be attributed to the components of PM10, including polycyclic aromatic
Acknowledgments
The authors express their gratitude to Yazmín Segura and Raúl Quintana, participants in the field campaign. This work was supported by CONACyT-Mexico Grants 43183-M, AC-2006-52830. Posdoctoral Fellowship to Yolanda I. Chirino from CONACyT (CVU 49870) and a Parker B. Francis Fellowship, USA, to Claudia María García-Cuellar are acknowledged. We thank Ingrid Mascher for revising the English version of the manuscript.
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These authors contributed equally to the work.